Collagen type III synthetic peptides for collagen resorption assays

ABSTRACT

Peptides synthesized to match C-telopeptide components of type III collagen degradation products in body fluids, selected from among EKAGGF (SEQ ID NO:39), IGGEKAGG (SEQ ID NO:40), IGGEKAGGF (SEQ ID NO:41), IAGIGGEKAGG (SEQ ID NO:42), IAGIGGEKAGGF (SEQ ID NO:43), and EKAGG (SEQ ID NO:44) are useful as immunogens and antigens in collagen resorption assays.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No. 10/009,999, filed Jul. 1, 2002 (now U.S. Pat. No. 6,602,980), which is the U.S. national phase of International Application No. PCT/US99/29357, filed Dec. 10, 1999, which is a continuation-in-part of U.S. application Ser. No. 09/385,740, filed Aug. 30, 1999 (now U.S. Pat. No. 6,348,320), which claims the benefit of U.S. Provisional Application No. 60/142,274, filed Jul. 2, 1999, and U.S. Provisional Application No. 60/141,574, filed Jun. 29, 1999, and is a continuation-in-part of U.S. application Ser. No. 09/335,098, filed Jun. 17, 1999 (now U.S. Pat. No. 6,255,056), which claims the benefit of U.S. Provisional Application No. 60/089,823, filed Jun. 19, 1998.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with government support under one or more of grants AR 37318 and AR 36794 awarded by the National Institutes of Health. The government has certain rights in the invention.

FIELD OF THE INVENTION

The invention relates to assays for detecting cross-linked telopeptide analytes indicative of type II collagen (cartilage) resorption in vivo, and in particular provides immunoassays for measuring and distinguishing between resorption of non-mineralized cartilage and mineralized cartilage, and for measuring total cartilage resorption in vivo.

BACKGROUND OF THE INVENTION

Reference is made to the applicant's prior U.S. Pat. Nos. 4,973,666, 5,140,103, 5,300,434, 5,320,970, 5,817,755, and 5,702,909, which are incorporated by reference herein.

Immunoassays are known for detecting telopeptide analytes indicative of collagen resorption in vivo. Examples of such type I collagen assays for measuring bone resorption include: WO 89/04491 (Eyre); WO 89/12824 (Robins); WO 91/08478 (Eyre); EP 0505210 A2 (Risteli & Risteli); WO 92/21698 (Eyre); WO 94/03813 (Naser et al.); WO 94/14844 (Baylink); WO 95/04282 (Naser et al.); WO 95/08115 (Qvist & Bonde); WO 96/12193 (Bonde & Qvist); EP 0718309 A1 (Naser et al.); and WO 96/36645 (Eyre et al.).

Examples of type II collagen telopeptide assays for measuring cartilage resorption include WO 91/08478 (Eyre); WO 95/08115 (Qvist & Bonde); and WO 96/12193 (Bonde & Qvist).

Examples of type III collagen telopeptide assays include WO 88/08980 (Risteli & Risteli) and WO 91/08478 (Eyre).

The following patent disclosures are also of interest: U.S. Pat. No. 4,628,027 (Gay) discloses in vitro diagnostic methods using monoclonal antibodies against connective tissue proteins. U.S. Pat. No. 5,316,914 (Oshima et al.) discloses an enzyme sandwich immunoassay of human type III, IV, and VI collagens applicable to diagnosis of hepatic diseases. WO 94/14070 (Poole & Hollander) discloses an immunoassay for the measurement of collagen cleavage in cartilage. WO 94/18563 (Barrach et al.) discloses sandwich immunoassay methods for detecting type II collagen derived peptides associated with arthritis.

Of particular interest is the applicant's prior international publication, WO 91/0478, which discloses the following urinary resorption products of type II collagen:

          17C       Glu-Hyl-Gly-Pro-Asp-(Pro)-(Leu) α1(II)C-telo-            | peptide       Glu-Hyl-Gly-Pro-Asp-(Pro)-(Leu) α1(II)C-telo-            | peptide (Gly)-Val-Hyl α1(II) heli-            87 cal domain wherein the parentheses indicate optional amino acid residues, and the cross-linking residue depicted as Hyl-Hyl-Hyl is hydroxylysyl pyridinoline (HP), a natural 3-hydroxypyridinium residue present in mature collagen fibrils of various tissues. These analytes derive from the C-terminal cross-linked telopeptide domain of type II collagen and so are collectively referred to herein as “col2CTx”.

The present disclosure also adopts the one-letter symbol form of amino acid shorthand; thus, the Glu-Hyl-Gly-Pro-Asp-(Pro)-(Leu) telopeptide components of the analytes shown above are referred to hereinafter as EKGPD (SEQ ID NO:1), EKGPDP (SEQ ID NO:2), and EKGPDPL (SEQ ID NO:3). Furthermore, as used herein the symbol “K” represents either lysine, in the case of linear peptides, or a cross-linking 3-hydroxypyridinium residue selected from among hydroxylysyl pyridinoline (HP) and lysyl pyridinoline (LP).

The applicant and colleagues have also described col2CTx in recent abstracts:

Eyre et al., Bone Miner. Res. 11(S1): S413, 1996, describes cross-linked telopeptides from collagen types I, II, and III in human urine.

Atley et al., Arth. Rheum. 40(9S):584, 1997, reports that RS-130830, a selective inhibitor of collagenase-3, blocks the release of hydroxyproline and a metalloproteinase (MMP) specific neoepitope, col2CTx, from bovine cartilage exposed to IL-1α.

Atley et al., Trans. Orthop. Res. Soc., New Orleans, 1998, reports matrix metalloproteinase-mediated release of immunoreactive telopeptides from cartilage type II collagen. The aim of this study was to evaluate whether an immunoassay based on a monoclonal antibody (mAb) 2B4, which recognizes a domain of the α1(II) C-telopeptide EKGPDP, measures MMP cleavage products in cartilage. Referring to FIG. 1, mAb 2B4 recognizes the in vitro product of matrilysin digestion AFAGLGPREKGPDP (SEQ ID NO:4) of synthetic peptide AFAGLGPREKGPDPLQYMRA (SEQ ID NO:5), but not AFAGLGPREKGPDPLQ (SEQ ID NO:6), AFAGLGPREKGPDPLQY (SEQ ID NO:7), LQYMRA (SEQ ID NO:8), or YMRA (SEQ ID NO:9). The authors also noted detection of mAb 2B4 immunoreactivity in synovial fluid, serum, and urine. They concluded that this 2B4 epitope has the potential to be a useful marker of type II collagen resorption in vivo.

Moskowitz et al., Amer. Coll. Rheum., San Diego, Calif., Nov. 8-12, 1998, reports that the type II collagen C-telopeptide 2B4 epitope is a marker for cartilage resorption in familial osteoarthrosis.

Eyre et al., 6^(th) International Meeting on Chemistry and Biology of Mineralized Tissues, Vittel, France, September 1998, discusses biochemical markers of bone and collagen degradation, including a cross-linked telopeptide product of type II collagen degradation in urine (col2CTx).

Atley et al., Third Combined Orthopaedic Research Societies Meeting, Hamamatsu, Japan, September 1998, discusses collagen type II cross-linked telopeptides, a promising marker for cartilage degradation in arthritis.

In addition, monoclonal antibodies to the C-telopeptide of type II collagen as diagnostic markers for rheumatoid arthritis are reportedly under development at Osteometer Biotech A/S. BioWorld International, page 3, Dec. 3, 1997.

The entire disclosures of the prior scientific and patent publications cited in this patent application are incorporated by reference herein.

SUMMARY OF THE INVENTION

The invention provides improved assays for measuring type II collagen (cartilage) resorption in vivo. The subject immunoassays are useful for distinguishing between resorption of non-mineralized cartilage and mineralized cartilage, and for measuring total cartilage resorption in vivo. The disclosed immunoassays employ either one or a combination of two antibodies. A first antibody binds to DEKAGGA (SEQ ID NO:21) but not to GGFDEKAGGAQLG (SEQ ID NO:27), where the K symbols refer to lysine residues and/or preferably to cross-linking 3-hydroxypyridinium residues. Measurement of analyte binding to the first antibody in serum provides an indication of non-mineralized cartilage resorption in vivo.

A second antibody binds to GGFDEKAGGAQLG but not to DEKAGGA, where the K symbols also preferably refer to cross-linking 3-hydroxypyridinium residues. Measurement of analyte binding to the second antibody in serum provides an indication of mineralized cartilage resorption in vivo.

An indication of total (non-mineralized and mineralized) cartilage resorption in vivo is provided by either measurement of analyte binding to the first antibody in urine, or by measurement of the total analyte binding to the first and second antibodies in serum.

The subject cartilage markers are preferably measured in conjunction with bone collagen resorption markers selected from among amino-terminal telopeptides of type I collagen, carboxy-terminal telopeptides of type I collagen, and free lysyl pyridinoline (deoxypyridinoline) cross-links.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

By way of introduction, the monoclonal antibody 2B4 recognizes a C-terminal epitope in the sequence EKGPDP. The antibody requires the C-terminal proline to be free for any binding (i.e., Pro-COOH) and prefers K to be derivatized (e.g., cross-linked) through its side-chain (epsilon) amino group. The presence of extra amino acids C-terminal to the proline prevents mAb 2B4 binding to this peptide domain.

Various matrix metalloproteinases (MMPs) including collagenases (MMP1 and MMP13), gelatinases (MMP2 and 9), stromelysin (MMP3), and matrilysin (MMP7) can generate the mAb 2B4 epitope from cartilage type II collagen or a synthetic C-telopeptide domain (AFAGLGPREKGPDPLQYMRA). Matrilysin (MMP7) is the most potent based on in vitro studies with the recombinant enzymes.

Cathepsin K, however, is unable to cleave the P-L bond required for 2B4 epitope release, but generates a longer fragment, EKGPDPLQ (SEQ ID NO:10). This is significant because cathepsin K is a protease expressed essentially only by osteoclasts and is the key enzyme responsible for the resorption of the calcified collagen when osteoclasts resorb bone. Osteoclasts (or a very similar cell) are also responsible for degrading calcified cartilage. Calcified cartilage is formed transiently in the growth plates of growing animals and is also present in the adult skeleton at the interfaces between bone and cartilage in all types of joints. In arthritis, accelerated resorption and bony remodeling will result in extensive resorption of calcified cartilage by osteoclasts. This process can release into the bloodstream collagen type II C-telopeptides (EKGPDPLQ) that are not recognized by 2B4.

Since the urinary form of the type II collagen telopeptide analyte is EKGPDP or smaller, proteases in the kidney and/or liver remove substantially all of the C-terminal—LQ before release into the urine. Therefore, two sources of immunoreactive col2CTx are can be defined: (1) direct generation by chondrocytes activated to degrade their matrix collagen by MMPs, and (2) secondary generation in the kidney and/or liver from the circulating products of osteoclastic resorption of calcified cartilage.

In arthritis (rheumatoid arthritis (RA) and osteoarthritis (OA)) MMPs are known to be key agents in the destruction of the collagenous matrices of joint cartilages. In both forms of arthritis, bone structure is also greatly affected. In RA, osteoporosis is a common consequence both directly from the disease process itself through the effects of inflammatory cytokines and as a side effect of corticosteroid therapy. The accelerated bone resorption will be most pronounced in involved joints and will include removal of the calcified cartilage underlying joint surfaces. In OA, osteophytes (bony outgrowths) often feature prominently in the joint pathology. Here, too, calcified cartilage will be formed and then degraded (the latter by osteoclasts) as part of osteophyte growth. Therefore to monitor and manage both major forms of arthritis it would be desirable to differentially measure MMP-mediated and osteoclast-mediated (cathepsin K) collagen type II destruction. In blood or synovial fluid, measuring col2CTx (EKGPDP) can index the former activity, whereas measuring the osteoclast product (EKGPDPLQ) in blood can index the latter. In addition, urinary col2CTx can provide an index of total resorption (both non-mineralized and mineralized cartilage). Such urine assays preferably include the steps of determining the creatinine content of the urine sample and correlating the ratio of the detected binding of the antibody to the creatinine content in order to provide a urinary index of total type II collagen resorption independent of urine volume.

The subject differential measurements of EKGPDP and EKGPDPLQ immunoreactivities have diagnostic value in the assessment of disease activity in the individual patient. In combination with measurement of a type I collagen marker to assess total bone resorption activity, and/or measurement of a type III collagen marker to assess other sources of non-mineralized connective tissue breakdown, an appropriate therapy can be prescribed and its desired effect monitored. Similarly, such differential assays can be used to identify new drug candidates (in vitro and in animals) and to provide surrogate markers in clinical trials for demonstrating desired beneficial effects on target tissues and cellular processes.

Representative assays for type I collagen resorption markers for this purpose include amino-terminal telopeptides of type I collagen (Osteomark®, Ostex International, Seattle, Wash.), carboxy-terminal telopeptides of type I collagen (CrossLaps®, Osteometer Biotech, Herlev, Denmark), and free lysyl pyridinoline cross-links (Pyrilinks®-D, Metra Biosystems, Mountain View, Calif.).

Representative assays for type III collagen resorption markers include amino-terminal telopeptides of type III collagen and carboxy-terminal telopeptides of type III collagen. Such assays are disclosed in U.S. Pat. No. 5,532,169, U.S. Pat. No. 5,641,687, and U.S. Ser. No. 08/923,175. Type III collagen resorption markers can be used to assess vascular collagen degradation (atherosclerosis), lung tissue degradation (e.g., emphysema and other destructive lung diseases), muscle wastage, frailty syndromes of the elderly, liver disease, hyperthyroidism, secondary effects of diabetes on connective tissue, and other inflammatory and infectious conditions that accelerate total bone resorption activity.

The invention also provides antibodies directed against other cross-linked carboxy-telopeptide degradation products of type II collagen, as well as linear peptides (SEQ ID NOS 11-20) synthesized to match the telopeptide components of the degradation products, and assays for cartilage degradation using the antibodies and linear peptides.

SEQ Body Fluid¹ α1(II)C Telopeptide ID Synovial Sequence NO fluid Blood Urine              EKGPD 1 — N N + M              EKGPDP 2 N N N + M              EKGPDPL 3 N N N + M              EKGPDPLQ 10 — M —          LGPREKGPDP 11 N N —          LGPREKGPDPLQ 12 N N —          LGPREKGPDPLQY 13 N N —       FAGLGPREKGPDP 14 N N —       FAGLGPREKGPDPLQ 15 N N —       FAGLGPREKGPDPLQY 16 N N — IDMSAFAGLGPREKGPDP 17 N N — IDMSAFAGLGPREKGPDPLQ 18 N N — IDMSAFAGLGPREKGPDPLQY 19 N N —             EKGPDPLQYMR 20 N N — ¹Cartilage Source: N = non-mineralized cartilage; M = mineralized cartilage; N + M = total cartilage degradation; and — = absent or only trace amounts.

The principal col2CTx degradation product is cross-linked EKGPDP. The further degradation product cross-linked EKGPD is usually present in urine in lower amounts.

The cross-linked EKGPDPL degradation product is a minor component relative to cross-linked EKGPDP.

Higher than normal levels of cross-linked EKGPDPLQ in blood can indicate mineralized cartilage degradation associated with active osteophyte formation in osteoarthritis.

The telopeptide sequences represented by SEQ ID NOS 11-13 result from metalloproteinase (MMP) cleavage of the G-L bond at the amino end of the α1(II)C telopeptide (SEQ ID NO 5), in combination with MMP cuts of the P-L, Q-Y, or Y-M bonds at the carboxy end of the telopeptide. SEQ ID NOS 14-16 represent related telopeptides from MMP cleavage between the A-F bond. SEQ ID NOS 17-19 represent longer telopeptides from MMP cleavage between the G-I bond. The amino ends of these longer telopeptides are generally metabolized further in the lymph nodes, liver, and/or kidney.

The telopeptide sequence represented by SEQ ID NO 20 results from serine protease cleavage of the R-E bond amino terminal to the cross-linking K residue, in combination with serine protease cleavage of the R-A bond at the carboxy end of the telopeptide.

Such cross-linking degradation products of SEQ ID NOS 11-19 (wherein the two telopeptide components are independently selected from among SEQ ID NOS 11-19), and SEQ ID NO 20, generally result from degradation of non-mineralized cartilage and are primarily found in synovial fluid and blood, and to a lesser extent in urine. Higher than normal levels of these degradation products in body fluids indicate active cartilage degradation that may correlate with rheumatoid arthritis, osteoarthritis, and other arthritides.

The invention also provides antibodies directed against cross-linked amino-telopeptide degradation products of type II collagen, as well as linear peptides (SEQ ID NOS 21-31) synthesized to match the telopeptide components of the degradation products, and assays for cartilage degradation using the antibodies and linear peptides.

Body Fluid² α1(II)N Telopeptide SEQ ID Synovial Sequence¹ NO fluid Blood Urine       DEKAGGA 21 N N N + M       DEKAGGAQ 22 N N N + M       DEKAGGAQL 23 N N N + M      FDEKAGGA 24 N N N + M      FDEKAGGAQ 25 N N N + M      FDEKAGGAQL 26 N N N + M    GGFDEKAGGAQLG 27 — M — JMAGGFDEKAGGAQLG 28 — M — JMAGGFDEKAGGAQLGV 29 N N —      FDEKAGGAQLGV 30 N N —       DEKAGGAQLGV 31 N N — ¹J = pyroglutamic acid, i.e., wholly cyclized pyrrolidone carboxylic acid (5-oxo-2-pyrrolidinecarboxylic acid). ²Cartilage Source: N = non-mineralized cartilage; M = mineralized cartilage; N + M = total cartilage degradation; and — = absent or only trace amounts.

The telopeptide sequences represented by SEQ ID NOS 21-23 result from metalloproteinase (MMP) cleavage of the F-Dbond at the amino end of the α1(II)N telopeptide, in combination with MMP cuts of the A-Q, Q-L, or L-G bonds at the carboxy end of the telopeptide. SEQ ID NOS 24-26 represent related telopeptides from MMP cleavage between the G-F bond at the amino end. The amino and carboxy ends of the longer telopeptides (SEQ ID NOS 22-26) are generally metabolized further in the lymph nodes, liver, and/or kidney. As a result, the principal col2NTx degradation products in urine are cross-linked DEKAGGA and FDEKAGGA.

The telopeptide sequences represented by SEQ ID NOS 27-28 result from cathepsin K cleavage of the G-V bond at the carboxy end of the telopeptide, in combination with cathepsin K cleavage of the A-G bond at the amino end of the telopeptide in the case of SEQ ID NO 27. In the case of SEQ ID NO 28 the amino terminal J residue is blocked. Higher than normal levels of cross-linked GGFDEKAGGAQLG and QMAGGFDEKAGGAQLG in blood can indicate mineralized cartilage degradation associated with active osteophyte formation in osteoarthritis. Proteases in the kidney and/or liver remove the carboxy-terminal G residue before release into the urine.

The telopeptides represented by SEQ ID NOS 29-31 result from metalloproteinase cleavage of the V-M bond at the carboxy end of the telopeptide, in combination with MMP cleavage of the G-F or F-D bond at the amino end of the telopeptide in the case of SEQ ID NOS 30-31. Larger degradation products comprising SEQ ID NOS 29-31 plus some additional helical sequence(s) may also be present in synovial fluid and blood. These cross-linked telopeptides are generally metabolized further in the lymph nodes, liver, and/or kidney, and consequently occur only in trace amounts in urine.

As indicated above, cross-linked α1(II)N telopeptides represented by SEQ ID NOS 27-31 are produced at the site(s) of cartilage degradation but degraded further during passage through the lymph nodes, liver, and/or kidney. The resulting smaller degradation products have two telopeptide components that may be independently selected from among SEQ ID NOS 21-26. Higher than normal levels of these degradation products in urine indicate total cartilage degradation that may correlate with rheumatoid arthritis, osteoarthritis, and other arthritides.

Thus the invention provides, in a first embodiment, an improved method of analyzing a body fluid sample for the presence of an analyte indicative of a physiological condition, including the steps of contacting the body fluid sample with an antibody which binds to the analyte, detecting binding of the antibody in the body fluid sample, and correlating any detected binding to the physiological condition, the improvement comprising contacting a serum sample with an antibody which binds to

      D-E-K-A-G-G-A           |       D-E-K-A-G-G-A           |           K but not to G-G-F-D-E-K-A-G-G-A-Q-L-G           | G-G-F-D-E-K-A-G-G-A-Q-L-G           |           K wherein K-K-K is hydroxylysyl pyridinoline or lysyl pyridinoline, and correlating any detected binding to resorption of non-mineralized type II collagen in vivo.

In a second embodiment, the invention provides an improved method of analyzing a body fluid sample for the presence of an analyte indicative of a physiological condition, including the steps of contacting the body fluid sample with an antibody which binds to the analyte, detecting binding of the antibody in the body fluid sample, and correlating any detected binding to the physiological condition, the improvement comprising contacting a serum sample with an antibody which binds to

G-G-F-D-E-K-A-G-G-A-Q-L-G           | G-G-F-D-E-K-A-G-G-A-Q-L-G           |           K but not to       D-E-K-A-G-G-A           |       D-E-K-A-G-G-A           |           K wherein K-K-K is hydroxylysyl pyridinoline or lysyl pyridinoline, and correlating any detected binding to resorption of mineralized type II collagen in vivo.

In a third embodiment, the invention provides an improved method of analyzing a body fluid sample for the presence of an analyte indicative of a physiological condition, including the steps of contacting the body fluid sample with an antibody which binds to the analyte, detecting binding of the antibody in the body fluid sample, and correlating any detected binding to the physiological condition, the improvement comprising contacting a urine sample with an antibody which binds to

      D-E-K-A-G-G-A           |       D-E-K-A-G-G-A           |           K but not to G-G-F-D-E-K-A-G-G-A-Q-L-G           | G-G-F-D-E-K-A-G-G-A-Q-L-G           |           K wherein K-K-K is hydroxylysyl pyridinoline or lysyl pyridinoline, and correlating any detected binding to resorption of both non-mineralized and mineralized type II collagen in vivo.

In a fourth embodiment, the invention provides an improved method of analyzing a body fluid sample for the presence of an analyte indicative of a physiological condition, including the steps of contacting the body fluid sample with an antibody which binds to the analyte, detecting binding of the antibody in the body fluid sample, and correlating any detected binding to the physiological condition, the improvement comprising contacting a serum sample with a first antibody which binds to

G-G-F-D-E-K-A-G-G-A-Q-L-G           | G-G-F-D-E-K-A-G-G-A-Q-L-G           |           K but not to       D-E-K-A-G-G-A           |       D-E-K-A-G-G-A           |           K and contacting the serum sample with a second antibody which binds to

      D-E-K-A-G-G-A           |       D-E-K-A-G-G-A           |           K but not to G-G-F-D-E-K-A-G-G-A-Q-L-G           | G-G-F-D-E-K-A-G-G-A-Q-L-G           |           K wherein K-K-K is hydroxylysyl pyridinoline or lysyl pyridinoline, and correlating the total detected binding of the first and second antibodies to resorption of both mineralized and non-mineralized type II collagen in vivo.

In a fifth embodiment, the invention provides an improved method of analyzing a body fluid sample for the presence of an analyte indicative of a physiological condition, including the steps of contacting the body fluid sample with an antibody which binds to the analyte, detecting binding of the antibody in the body fluid sample, and correlating any detected binding to the physiological condition, the improvement comprising contacting a serum sample with a first antibody which binds to

G-G-F-D-E-K-A-G-G-A-Q-L-G           | G-G-F-D-E-K-A-G-G-A-Q-L-G           |           K but not to       D-E-K-A-G-G-A           |       D-E-K-A-G-G-A           |           K and contacting a urine sample with a second antibody which binds to

      D-E-K-A-G-G-A           |       D-E-K-A-G-G-A           |           K but not to G-G-F-D-E-K-A-G-G-A-Q-L-G           | G-G-F-D-E-K-A-G-G-A-Q-L-G           |           K wherein K-K-K is hydroxylysyl pyridinoline or lysyl pyridinoline, and correlating any detected binding of the first antibody to resorption of mineralized type II collagen in vivo, and any detected binding of the second antibody to resorption of both mineralized and non-mineralized type II collagen in vivo.

The term “antibody” in this disclosure is meant to encompass polyclonal antibodies, monoclonal antibodies, and antigen-binding fragments thereof. Such EKGPDPLQ-binding and EKGPDP-binding antibodies, or GGFDEKAGGAQLG-binding and DEKAGGA-binding antibodies, can be produced by standard methods well known in the art. A representative protocol follows.

EKGPDPLQ hybridoma development: Female RFB/DnJ mice are injected intraperitoneal (ip) with an emulsion of complete Freund's adjuvant and EKGPDPLQ synthetic peptide cross-linked to the carrier protein keyhole limpet hemacyanin (KLH) via glutaraldehyde (KLH-EKGPDPLQ). One month later a booster injection of KLH-EKGPDPLQ is given ip in an emulsion with incomplete Freund's adjuvant. Approximately eight weeks later the mice are sacrificed and splenocytes fused, by polyethylene glycol, to the FOX-NY murine myeloma cell line (ATCC CRL-1732). Fusion cells are grown in 96-well plates in selection media containing adenine, aminopterin and thymidine (AAT).

Approximately one week after fusion hybridoma colony culture supernatants are tested in ELISA for reactivity to the EKGPDPLQ synthetic peptide cross-linked via glutaraldehyde to bovine serum albumin (BSA-EKGPDPLQ). Hybridoma colonies producing antibody reactive with BSA-EKGPDPLQ are secondarily tested for specificity of reaction in ELISA against other synthetic peptide sequences. Among those tested are EKGPDP synthetic peptide cross-linked to BSA via glutaraldehyde (BSA-EKGPDP). Hybridoma colonies that react positively to BSA-EKGPDPLQ and negatively to BSA-EKGPDP are cryopreserved. Representative hybridomas are cloned to monoclonality by the limited dilution method.

In competition ELISA, human serum specimens as well as soluble BSA-EKGPDPLQ are found to compete with solid phase BSA-EKGPDPLQ for candidate antibodies.

A cathepsin K digest of type II collagen can also be prepared for immunization and screening purposes. Proteoglycans are extracted from a tissue sample of human articular cartilage with a protein denaturant, such as 4 M guanidine HCl. The residue is washed with water and digested with recombinant cathepsin K (37° C., pH 5.8). The digest is fractionated by high performance liquid chromatography, for example by reverse phase, and fractions containing pyridinoline cross-links are monitored by fluorescence emission at 390 nm (297 nm excitation). The fluorescent fraction containing the cross-linked C-telopeptides of sequence EKGPDPLQ is identified by N-terminal sequence analysis. This fraction can be used for screening purposes and for validation of antibody specificity.

The subject antibodies can be employed in a variety of immunoassay formats that are well known in the art. For example, see: Harlow & Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, pp. 553-612, 1988; Principles and Practice of Immunoassay, Price & Newman (Eds.), Stockton Press, 1991; and Immunoassay, Diamandis & Christopoulos (Eds.), Academic Press, 1996.

Type III Collagen

This invention provides antibodies directed against cross-linked amino-telopeptide degradation products of type III collagen, as well as linear peptides (SEQ ID NOS 33-37) synthesized to match the telopeptide components of the degradation products, and assays for type III collagen degradation using the antibodies and linear peptides. For reference, the native α1(III)N telopeptide has the following amino acid sequence: JYDSYDVKSGVAVGGLAG (SEQ ID NO 32).

α1(III)N Telopeptide SEQ Synovial Sequence¹ ID NO fluid Blood Urine         DVKSGVAVGG 33 x x x        YDVKSGVAVGG 34 x x x       SYDVKSGVAVGG 35 x x x      DSYDVKSGVAVGG 36 x x x    JYDSYDVKSGVAVGG 37 x x x proQYDSYDVKSGVAVGG 38 x x x ¹J = pyroglutamic acid, i.e., wholly cyclized pyrrolidone carboxylic acid (5-oxo-2-pyrrolidinecaiboxylic acid). ²x = present.

The preferential site for initial metalloproteinase (MMP) cleavage is the G-L bond near the carboxy end of the α1(III)N telopeptide. Thus, the cross-linked col3NTx degradation products in body fluids all share the distinctive -KSGVAVGG neoepitope.

The telopeptides represented by SEQ ID NOS 33-36 result, for example, from metalloproteinase (MMP) cleavage of the Y-D, S-Y, D-S, or Y-D bonds at the amino end of the telopeptide, in combination with the G-L cleavage at the carboxy end.

The telopeptide represented by SEQ ID NO 37 retains the amino terminal J residue. Larger variants have the intact globular N-propeptide, or a fragment thereof, still retained to the amino terminus (wherein the terminal residue is Q). These larger variants are collectively indicated in the Table above as “proQYDSYDVKSGVAVGG”. Even these largest forms containing N-propeptide remnants pass into urine. All are preferably assayed using an antibody directed against the cross-linked-KSGVAVGG epitope. Assay for these fragments by this preferred epitope provides an index of structural type III collagen degradation rather than new synthesis.

This invention also provides antibodies directed against cross-linked carboxy-telopeptide degradation products of type III collagen, as well as linear peptides (SEQ ID NOS 39-44) synthesized to match the telopeptide components of the degradation products, and assays for type III collagen degradation using the antibodies and linear peptides. For reference, the native α1(III)C telopeptide has the following amino acid sequence: GGVGAAAIAGIGGEKAGGFAPYY (SEQ ID NO 38).

Body Fluid¹ α1(III)C Telopeptide SEQ Synovial Sequence ID NO fluid Blood Urine       EKAGGF 39 — — x    IGGEKAGG 40 x x x    IGGEKAGGF 41 x x — IAGIGGEKAGG 42 x x — IAGIGGEKAGGF 43 x x — ¹x = present; and — = absent or only trace amounts.

The preferential site for initial metalloproteinase (MMP) cleavage is the A-I bond near the amino end of the α1(III)C telopeptide. The telopeptides represented by SEQ ID NOS 43 and 42 result from MMP cleavage of the F-A or G-F bonds at the carboxy end of the telopeptide, in combination with the A-I cleavage at the amino end. The telopeptide represented by SEQ ID NO 41 results from further processing by MMP cleavage of the G-I bond at the amino end of the telopeptide. These degradation products are present in blood and synovial fluid but are absent or found only in trace amounts in urine.

Additional cleavages in the kidney result in smaller fragments in the urine. The telopeptide represented by SEQ ID NO 40 results from MMP cleavage of the G-I bond at the amino end of the telopeptide, in combination with the G-F cleavage at the carboxy end. This cross-linked degradation product is found in all body fluids.

The telopeptide represented by SEQ ID NO 39 results from cleavage by kidney proteases of the G-E bond at the amino end of the telopeptide. This cross-linked degradation product is therefore found in significant amounts only in urine. Even smaller cross-linked peptides, including those with telopeptide component(s) EKAGG (SEQ ID NO 44), also appear in urine through the action of proteases in the kidney.

While the invention has been described in conjunction with preferred embodiments, one of ordinary skill after reading the foregoing specification will be able to effect various changes, substitutions of equivalents, and alterations to the subject matter set forth herein. Hence, the invention can be practiced in ways other than those specifically described herein. 

1. A synthetic peptide selected from among EKAGGF (SEQ ID NO:39), IGGEKAGG (SEQ ID NO:40), IGGEKAGGF (SEQ ID NO:41), IAGIGGEKAGG (SEQ ID NO:42), IAGIGGEKAGGF (SEQ ID NO:43), and EKAGG (SEQ ID NO:44). 